The viridans streptococci are a group of gram-positive bacteria that constitute part of the resident flora in the human oral cavity and gastrointestinal tract. They can occasionally be found as a cause of transient bacteremia, especially following dental procedures. This group of streptococci has become one of the important causative agents of subacute bacterial endocarditis that can result in serious damage of heart valves. Viridans streptococci are composed of at least 22 species, which are currently divided into 5 subgroups: mitis, anginosus, mutans, salivarius, and bovis. Mitis group streptococci are the most common viridans streptococci responsible for diseases in humans. In the past decades, with the emergence of molecular-based methods, re-classification of viridans streptococci has been evolving and is sometimes confusing. One of the reasons for the ambiguity is that conventional biochemical tests used to identify most of the bacteria isolated in the clinical laboratory often do not provide definitive identification at the species level, and may also misidentify members of this group. A new approach for the identification of bacterial pathogens using 16S rDNA gene sequences has been recently introduced. However, this technique has not been helpful in some closely related organisms that have nearly identical sequences in their 16S genes. The 16S rDNA sequencing approach for identification of clinically important bacteria, however, has real limitations. We decided to try the multilocus gene sequencing targets-- housekeeping genes --for differentiating bacteria at the species level, even though this molecular version of starch gel protein electrophoresis has not been widely used for species differentiation. We set out to try this approach for species delineation of bacterial isolates belonging to the mitis and sanguinis groups of viridans streptococci. Although logically the finer discriminations required for purposes of successful strain typing would suggest that this approach might give cleaner distinctions between organisms at the species level, it was unknown whether or not conserved oligonuceotides for PCR amplification of targeted gene regions could be found for all the species of these two groups; or whether any sequence differences detected in house keeping genes in these unexamined species would reliably differentiate organisms at the species level or prove to be just random mutations that are stably present for short evolutionary time periods and, thereby, only useful at distinguishing bacteria at the stain level. We were able to demonstrate that two of the housekeeping gene targets used for S. pneumoniae strain typing could also be used for very clear species discriminations in these two clinically important groups of viridans streptococci. Our studies successfully demonstrated that two of the genes studied, gdh and gki, could be applied to species identification in these organisms. The discriminatory power of this approach is far better that 16S rDNA sequencing. This work has been described in a submitted manuscript.